DE19841299A1 - Novel gene for a variant Gbeta3-subunit of human G-protein useful for determining risk of G-protein dysfunction associated disease - Google Patents

Abstract

Nucleic acid sequences (I) encoding a G beta 3-subunit of the human G-protein comprising a 1523 base pair (bp) sequence (given in the specification), are new. An Independent claim is also included for a method of determining the risk of a patient developing a G-protein associated disease, by comparing the patients G beta 3-subunit gene sequence with the sequence of (I) and diagnosing an increased risk of disease if the sequences agree at positions 825 and/or 1429.

Description

The present invention relates to a new one Nucleic acid sequence coding for the Gβ3 subunit of human G protein, and the use of Gβ3 subunits the G proteins to determine the risk of an illness, associated with G protein misregistration fall ill.

Heterotrimeric guanine nucleotide binding proteins (G proteins) have a prominent role in intracellular Signal transduction. They mediate the forwarding extracellular signals after stimulation of hormone receptors and other receptors, which have a Go through conformation change. This leads to activation of G proteins, which are subsequently called intracellular effectors (e.g. tonal channels, enzymes) can activate or inhibit. G- Proteins regulate intracellular signal processing hormonal stimulation of heptahelical receptors in the Cell membrane, but also after stimulation of receptors with intrinsic tyrosine kinase activity. Regulated Cell functions include cell division and Cell growth, contraction, release of cell contents and much more

Heterotrimeric G proteins are made up of three subunits, the α-, β and γ subunits. So far several different α subunits, 5 β subunits and about 12 γ subunits using biochemical and proven molecular biological methods (Birnbaumer, L. and Birnbaumer, M., Signal transduction by G proteins: 1994 edition. J. Recept. Res. 15: 213-252, 1995; Offermans, S. and Schultz, G.  Complex information processing by the transmembrane signaling system involving G proteins. Naunyn Schmiedebergs Arch. Pharmacol. 350: 329-338, 1994; Nuremberg, B., Gudermann, T. and Schultz, G. Receptors and G proteins as primary components of transmembrane signal transduction. Part 2. G proteins: structure and function. J. Mol. Med. 73: 123-132, 1995; Neer, E.J. Heterotrimeric G proteins: Organizers of Transmembrane Signals. Cell 80: 249-257, 1995; Rens-Domiano, S. and Hamm, H.E. Structural and functional relationships of heterotrimeric G proteins. FASEB J. 9: 1059-1066, 1995).

The receptor-mediated activation of certain α subunits can be pretreated with pertussis toxin (PTX) can be inhibited. These include in particular the α- Isoforms α-i1, α-i2 and α-i3 as well as different αo- Subunits. Such G proteins are also called "PTX called sensitive G proteins.

βγ subunits perform essential functions in the G protein activation as well as in the modulation of intracellular Reactions. All previously known G protein β subunits point at the level of the nucleotide sequence and at the level high homologies in the amino acid sequence. Doing this Similarities not only within the human β subunits (Gβ1, Gβ2, Gβ3) found, but also compared to β subunits of other species, for example fruit flies or yeast.

Recently, in the human GNB3 gene, which is responsible for the Gβ3- Subunit encoded, a base change in exon 10 (C825T) described for alternative splicing of exon 9 leads. The alternative splicing is done by a cryptic Favored splice in exon 9, with the distant one Base exchange C825T reinforces the splicing. The alternative Splice product (Gβ3s) shows a loss of 123 bp (= 41 Amino acids). The GNB3-825T allele is with increased  Activation of G proteins and essential hypertension associated (Siffert, W., Rosskopf, D., Siffert, G., Busch, S., Moritz, A., Erbel, R., Sharma, A.M., Ritz, E., Wichmann, H.E., Jakobs, K.H., and Horsthemke, B. Association of a human G- protein beta 3 subunit variant with hypertension. Nat.Genet. 18 (1): 45-48, 1998; Clapham, D.E. and Neer, E.J. G protein βγ subunits. Annu. Rev. Pharmacol. Toxicol. 37: 167-203, 1997; Hamm, H.E. and Gilchrist, A. Heterotrimeric G proteins. Curr. Opin. Cell Biol. 8: 189-196, 1996).

This human GNB3 gene was developed by Levine et al. (Levine, M.A., Smallwood, P.M., Moen, P.T. Jr., Helman, L.J. and Ahn, T.G. Molecular cloning of beta 3 subunit, a third form of the G protein beta subunit polypeptides. Proc. Natl. Acad. Sci. UNITED STATES. 87 (6), 2329-2333 (1990)).

The invention relates to a new human cDNA for the Gβ3- Subunit of human G protein.

Surprisingly, it turned out that it was there is another polymorphism in the human GNB3 gene which hereinafter called "C1429T". This polymorphism takes place at position 1429 of the cDNA. This corresponds to exon 11 of the pre-mRNA, but not outside the open reading frame in 3 ' translated area.

This polymorphism is pronounced Distribution imbalance with the well-known C825T Polymorphism, such that almost all GBN3-825C alleles Genotype 1429C and almost all GBN3-825T alleles the genotype 1429T. This polymorphism C1429T is therefore suitable as well as the polymorphism C825T for the detection of increased activation of G proteins.

The polymorphism C1429T is based on the sequence of the cDNA as described by Levine and co-workers. At position 1429 of the cDNA, the C is replaced by a T:

The complete cDNA sequence is listed in Appendix 1.

Based on the genomic sequence of the GBN3 locus as described by Ansari-Lari and co-workers (Ansari-Lari, MA, Muzny, DM, Lu, J., Lu, F., Lilley, CE, Spanos, S., Malley , T. and Gibbs, RA, A gene-rich cluster between the CD4 and triosephosphate isomerase genes at human chromosome 12p13. Genome Res. 6 (4), 314-326 (1996)), this polymorphism is localized as follows (C59308T) :

In Appendix 2 is the complete genomic sequence listed.

This polymorphism is verified by a person skilled in the art common methods such as specific hybridization, Sequencing, PCR reaction with subsequent Restriction analysis, DNA chip technology, single strand conformation polymorphism etc. In an exemplary experiment detection was carried out by amplification of the corresponding Gene segment and subsequent analysis of the restriction Fragment length polymorphism in which the restriction enzymes BanI, BshNI, Eco64I or their isoschizomers can be used.  

As a result, the genotype at positions 825 and 1429 is very similar:

The new nucleic acid sequence can be used to produce Antisense drugs for therapy or prevention of Diseases serve, being one to this nucleic acid sequence complementary nucleic acid sequence for the production of the antisense Drug is used. The patients can z. B. with antisense oligonucleotides or vectors for prevention the transcription or translation of the Gβ3 subunit be treated.

The invention further relates to the use of β3- Subunits of G proteins to determine the risk a disease associated with G protein misregistration is associated to become ill.

Although the Gβ3s splice variant, which has the polymorphism C1429T or is due to the C825T polymorphism, in combination with the G protein subunits Gαi2 and Gγ5 can form a functional heterotrimer, it was unclear on which way Gβ3s leads to an increased activation of G- Proteins.

The object of the present invention is based on the knowledge that the subunit Gβ3s leads to an increased activatability of G proteins. The increased activatability of G proteins was demonstrated by transfection of the corresponding cDNAs and expression of Gβ3 and Gß3s in the COS-7 transfection system which is well known to the person skilled in the art. Here it was shown that the activatability of G proteins after transfection of Gß3s compared to Gβ3 is significantly increased ( FIG. 1). To quantify the activatability of G proteins, the incorporation of radioactively labeled [35S] GTPγS in G protein α subunits was measured after stimulation with the peptide Mastoparan-7 (Mas-7).

Similar results can be achieved in the Sf9 insect cell expression system ( FIG. 2). Here the m ² muscarinic receptor is expressed together with the G protein subunits Gαi3 and Gγ5 and either Gβ3 or Gβ3-s in the Sf9 system. After stimulation with the agonist carbachol, an increased potency and an increased efficiency of the agonist carbachol in relation to the stimulability of G proteins are observed in the presence of Gβ3-s.

These experiments show that the splice variant Gβ3-s for one Caused increased activation of G proteins responsible for. Furthermore, these experiments prove that the Protein is a gene therapy for diseases associated with are associated with such a G protein misregistration, in the sense suitable for achieving increased cellular reactivity.

Among diseases with G protein misregistration are associated, such diseases are to be understood at which the G protein is involved in signal transduction and does not perform its function in a physiological manner. The Mismanagement can have a number of causes. for example a change in the structural gene or a altered gene expression. The present invention relates to Diseases associated with the GNB3-825T allele described above or GNB3-1429T allele are associated. These include diabetes mellitus (type 2), obesity and obesity, coronary Heart disease, immune disorders due to an increased Function of the immune system, and high-risk pregnancies with the Risk of premature birth (premature birth).  

The following examples refer to tests with the GNB3-825T allele. Because of the great agreement (Coupling balance) between the genotype at positions 825 and 1429 are these examples and those from the Test Results Drawn Conclusions for the GNB3 1429T allele also valid.

1. Predicting Diabetes Mellitus (Type 2)

Type 2 diabetes (synonyms: adult diabetes, non-insulin diabetes) is a serious illness with high cardiovascular morbidity and mortality. Genetic Influences and obesity contribute significantly to the pathogenesis at. Type 2 diabetes often begins as insulin resistance, which is initially due to increased insulin secretion is compensated so that the affected individuals asymptomatic stay (euglycemic). Only when the increased Insulin secretion can no longer be maintained diabetes occurs with increased blood sugar levels. On cellular level can change in components of the Insulin signal transduction, e.g. B. with insulin receptor substrate 1 (IRS-1), PI-3 kinases, protein kinases, etc. one Cause insulin resistance. But also after one Cell stimulation with agonists (e.g. angiotensin II), the G- Activating protein-coupled receptors can be a cellular Insulin resistance can be brought about. The effect of a subsequent stimulation with insulin is then clear reduced (Polonsky, K.S., Sturis, J., and Beil, G.I. Non insulin-dependent diabetes mellitus - A genetically programmed failure of the beta cell to compensate for insulin resistance. N. EngI. J. Med. 334: 777-783, 1996; O'Doherty, R., Stein, D., and Foley, J. Insulin resistance. Diabetologia 40 Suppl 3: B10- 5: B10-5, 1997; Kahn, C.R., Vicent, D., and Doria, A. Genetics of non-insulin-dependent (type-lI) diabetes mellitus. Annu. Rev. Med. 47: 509-531, 1996; Hansen, T., Andersen, C. B., Echwald, S.M., Urhammer, S.A., Clausen, J.O., Vestergaard, H., Owens, D., Hansen, L., and Pedersen, O. Identification of a  common amino acid polymorphism in the p85alpha regulatory subunit of phosphatidylinositol 3-kinase: effects on glucose disappearance constant, glucose effectiveness, and the insulin sensitivity index. Diabetes 46 (3): 494-501, 1997; Folli, F., Kahn, C.R., Hansen, H., Bouchie, J.L., and Feener, E.P. Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels - A potential role for serine phosphorylation in insulin / angiotensin II crosstalk. J. Clin. Invest. 100: 2158-2169, 1997; Zhang, Y., Wat, N., Stratton, I.M., Warren-Perry, M.G., Orho, M., Groop, L., and Turner, R.C. UKPDS 19: heterogeneity in NIDDM: separate contributions of IRS-1 and b3-adrenergic receptor mutations to insulin resistance and obesity respectively with no evidence for glycogen synthase gene mutations. Diabetologia 39: 1505-1511, 1996; Almind, K., Bjorbaek, C., Vestergaard, H., Hansen, T., Echwald, S., and Pedersen, O. Aminoacid polymorphisms of insulin receptor substrate-1 in non-insulin-dependent diabetes mellitus. Lancet 342: 828-832, 1993; Laakso, M., Malkki, M., Kekäläinen, P., Kuusisto, J., and Deeb, S. S. Insulin receptor substrate-1 variants in non-insulin-dependent diabetes. J. Clin. Invest. 94: 1141-1146, 1994).

This G protein activation leads to phosphorylation of IRS-1 on serine residues, resulting in that induced by insulin Phosphorylation on tyrosine residues is reduced. As a result this leads to a reduced interaction of IRS-1 with the insulin receptor and the PI-3 kinase, d. H. to a decreased insulin action. An increased activatability of G proteins by the GNB3 825T allele and thus connected splice variant Gβ3-s in vivo is strengthened the tendency to insulin resistance clearly.

It has now been shown that with the simultaneous presence of Mutations in components of insulin signal transduction (IRS1- Gene, 3931A variant; Gly971Arg; p85α regulatory Subunit of PI3 kinase (1020 G → A; codon 326 Met → Ile;  b3 adrenergic receptor (Trp64Arg); β2-adrenergic receptor (here especially the Arg16Gly variant and the Gln27Glu variant); Tumor necrosis factor α; Leptin or the leptin receptor) which lead to insulin resistance, and the GNB3-825T allele, which Tendency to insulin resistance and diabetes dramatically increases. This connection opens up the possibility of a Type 2 diabetes mellitus associated with the GNB3-825T allele diagnose and such a genetic bias for type 2 diabetes mellitus in healthy or to predict symptom-free people.

As evidence, the DNA of over 700 patients with type 2 Diabetes and obtained from 1400 healthy controls. The Frequencies of the GNB3-825T allele and the IRS-1 Gly971Arg Variant were compared. Table I first shows one Comparison of allele frequencies in controls and cases:

Table I

Allele frequencies in controls and type 2 diabetics

The following Tables IIa and IIb show the risk for carriers of the GNB3-825T allele or carriers of the IRS1-3931A allele (simple effects, Table II.1) or for carriers of both alleles (combined effects, Table II.2) to develop type 2 diabetes. The risk is expressed here as an age-adjusted odds ratio, with the odds ratio for case-control studies roughly corresponding to the relative risk in prospective studies.

You can see a clear increase in risk for type 2 Diabetes for carriers of the GNB3-825T allele or carrier of the IRS1 3931A alleles (Tables II.1 and II.2). A drastic one Risk increase is found in individuals with both Genes are changed. Thus the verification of the GNB3-825T- Alleles serve the inclination in the individuals concerned for a disease of type 2 diabetes or one genetic cause of an already occurring To determine disease.

2. Predict obesity / overweight

The regulation of the Body Mass Index (BMI), a measure of the Ratio of body weight to height is determined by many Genes determined. Overweight is about 40% genetic, is also due to excessive calorie intake in existing Lack of exercise causes. The Gβ3-s splice variant is included the cellular phenotype of increased cell growth connected. It is also possible that the GNB3-825T allele to an increased body growth, u. a. Overweight, predisposed, and can have epi and hypostatic effects. As with diabetes mellitus (type 2), one can Are related to changes in the IRS1 gene (3931A- Variant; Gly971Arg), in the gene for the β3-adrenergic gene Receptor coded (Trp64Arg variant) and in the gene that 2-adrenergic receptor coded, here in particular the Arg16Gly- Variant and the Gln27Glu variant.

Studies on transgenic mice showed that the absence of the In contrast, gene encoding the IRS-1 protein leads to a strong delay in body growth (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S. Insulin resistance  and growth retardation in mice lacking insulin receptor substrate-1. Nature 372: 182-186, 1994).

In order to check the correlation of obesity with the presence of the GNB3- 825 nucleotide and the status of the IRS1 gene with regard to the presence of the Gly971Arg variant, the BMI was obtained in 20-30 year old healthy men who also had the status of the GNB3 -825 nucleotide and the status of the IRS1 gene with respect to the presence of the Gly971Arg variant was measured. The result is shown in Fig. 3.

Fig. 3 shows the frequency distribution of the measured values for the BMI. In the presence of the more common IRS1 "GG" genotype, the GNB3-825T allele shows a significant tendency towards increased BMI compared to the GNB3-C825 allele. The odds ratio for the 75% quartile versus the 25% quartile is 2.5. In contrast, the tendency to increase BMI in carriers of the GNB3-825T allele in the presence of the IRS1-3931A allele (Gly971Arg variant) is clearly counteracted. This illustrates the interactive effect of GNB3 and IRS1 on the BMI. Thus, the presence of the GNB3-825T allele can be reliably correlated with obesity. This makes it possible to predict a tendency towards obesity in carriers of this allele, in particular those who simultaneously lack the IRS1-3931A allele (Gly971Arg variant).

3. Prediction of coronary artery disease and atherosclerosis

It is already known that coronary artery disease can be associated with G protein misregistration. In order to test a relationship between the occurrence of coronary artery disease and the presence of the GNB3-825T allele, patients with angiographically excluded coronary artery disease, with coronary artery disease (without myocardial infarction), with a myocardial infarction and with more than one myocardial infarction were examined for the presence of this Everything checked. The result is shown in Fig. 4.

Fig. 4 shows the frequency of the GNB3-825T allele in patients with angiographically excluded coronary artery disease (CHD), with CHD (without myocardial infarction; MI), with a myocardial infarction (MI) and with more than one myocardial infarction.

You can see a significant increase in the frequency of the GNB3- 825T alleles in CHD and MI. The risk for CHD and MI will be by the GNB3-825T allele compared to controls without CAD doubled.

Patients with a mutation in the IRS-1 protein (3931A allele; Gly971Arg variant), however, experience a clear one Risk reduction by up to 50% if the GNB3-C825- or the GNB3-825T allele. This change in the IRS-1 protein exerts hypostatic effects, i. H. this variant protects from coronary artery disease.

Comparing patients with CAD to individuals with coronary angiographically excluded CAD shows the following odds ratios:

This is a correlation between the existence of the GNB3 825T allele with the occurrence of coronary artery disease. Thus, it is possible to have a tendency to coronary artery disease in carriers of this allele, especially those whose at the same time the IRS1-3931A allele (Gly971Arg variant) is missing, to predict.

A special area of application is the prediction of a coronary disease, but also general of cardiovascular risk (high blood pressure, etc.) in women with the goal of this being a targeted, post-menopausal  To administer hormone therapy with female sex hormones reduce cardiovascular risk.

Another area of application is the prediction of an increased risk of myocardial infarction and sudden cardiac death. This is partly due to the fact that G proteins also control ion channels. More specifically, the Gα and Gßγ subunits of G proteins control the function of multiple ion channels, e.g. B. Na ⁺ channels, Ca ²⁺ channels and K ⁺ channels. A precisely coordinated regulation of such ion channels is of great importance for all electrically excitable tissues, especially for the heart (De Waard, M., Liu, H., Walker, D., Scott, VE, Gurnett, CA, and Campbell, KP Direct binding of G-protein βγ complex to voltage-dependent calcium channels. Nature 385 (6615): 446-450, 1997; Ma, JY, Catterall, WA, and Scheuer, T. Persistent sodium currents through brain sodium channels induced by G protein βγ subunits. Neuron 19 (2): 443-452, 1997; Kofuji, P., Davidson, N., and Lester, HA Evidence that neuronal G-protein-gated inwardly rectifiying K ⁺ channels are activated by Gβγ subunits and function as heteromultimers, Proc. Natl. Acad. Sci. USA 92: 6542-6546, 1995; Krapivinsky, G., Krapivinsky, L., Wickman, K., and Clapham, DE Gβγ binds directly to the G protein-gated K ⁺ channel , I _KACh . J. Biol. Chem. 270: 29059-29062, 1995).

It has been shown that persons who carry the GNB3-T825 allele show an increased activity of myocardial K ⁺ channels. This leads to an accelerated repolarization of the heart muscle cell, and thus to a shorter refractory period. These people are at increased risk of arrhythmias, especially ventricular tachycardias, extrasystoles, ventricular flutter and ventricular fibrillation. You are at increased risk of sudden cardiac death even as part of an acute myocardial infarction.

After all, carriers of the GNB3-825T allele show in old age from 20-30 years, significant changes in properties of blood vessels. An increased is particularly noticeable Pulse wave speed, an increased stroke volume of the Heart and an increased pulse pressure. These phenomena are Expression of an early tendency to an elevated Stiffness of the blood vessels (reduced compliance) as an indicator for atherosclerosis. The genotyping for determination the GNB3-C825T allele status is therefore suitable for an increased Determine risk of atherosclerosis.

4. Prediction of an enhanced function of the immune system

G proteins and G protein-coupled receptors are also found in all cells of the immune system, especially in Leukocytes. Chemotaxis of cells is mainly caused by βγ- Subunits of heterotrimeric G proteins mediated. In order to should also increase the GNB3-825T allele Responsiveness of the immune system, especially to one lead to increased immune defense.

Indeed, neutrophil granulocytes from carriers of the GNB3-825T allele show an increased chemotaxis compared to the peptide fMLP ( FIG. 5). fMLP is a peptide that is representative of a variety of bacterial peptides and stimulates chemotactic reactions. It therefore serves as a test system for measuring chemotactic reactions of cells, which is well known to the person skilled in the art. The fMLP receptor is known to activate pertussis toxin-sensitive G proteins. The finding that granulocytes from carriers of the GNB3-825T allele show an increased fMLP-stimulated chemotaxis is consistent with the fact that the chemotaxis is mediated by heterodimeric βγ subunits.

This phenomenon can also be found in other leukocytes, e.g. B. Detect lymphocytes. There is therefore a correlation between the GNB3-825T allele and an increased chemotaxis  of cells of the immune system, e.g. B. of neutrophils Granulocytes, T-lymphocytes or B-lymphocytes.

Furthermore, one is observed in carriers of the GNB3-825T allele increased proliferation tendency of cells of the immune system, especially after vaccinations.

Healthy carriers of the GNB3-825T allele show an increased number of leukocytes and of CD4-positive T-lymphocytes (absolute and percentage) with an increased CD4 / CD8 quotient. Figure 6 shows this for the increased number of CD4 lymphocytes. This can also be demonstrated in AIDS patients in whom the CD4 number indicates the severity of the disease or the expected lifespan ( FIG. 7). This is accompanied by an increased tendency to maintain an increased number of CD4-positive T lymphocytes despite HIV infection. Carriers of the GNB3-825T allele also show an increased tendency to contract AIDS after HIV infection, also in connection with the detection of the gene change described above in chemokine receptors, in particular a Δ32 deletion in the CCRK5 receptor or in the region of the promoter of this gene.

Finally, one is observed in the people affected increased release of immunomodulatory substances, Hormones and other substances from leukocytes (cytokines, Interleukins, growth factors, antibodies, vascular Substances). This also results in a increased immune defense after transplantation of organs or Tissues (kidney, heart, bone marrow, lungs, skin, liver etc.) with the risk of graft rejection. It also follows from this an increased tendency to autoimmune diseases (rheumatism, Ulcerative colitis, Crohn's disease) and allergic Diseases, e.g. B. the skin, respiratory tract or other organs (e.g. neurodermatitis, hay fever, bronchial asthma). This is also observed in combination with the detection of others Genetic changes, e.g. B. in the β2-adrenergic receptor, here especially the Arg16Gly variant and the Gln27Glu variant.  

5. Prediction of an increased CO ₂ or hypoxia tolerance

As is well known, G proteins are also involved in the regulation of Breathing drive involved through hypoxia or hypercapnia (Prabhakar, N.R., Kou, Y.R., and Kumar, G.K. G proteins in carotid body chemoreception. Biol. Signals 4: 271-276, 1995; Cachero, T.G., Rocher, A., Rigual, R.J., and Gonzalez, C. Effects of fluoride and cholera and pertussis toxins on sensory transduction in the carotid body. At the. J. Physiol. Cell Physiol. 269: C1271-C1279, 1995).

It has been found that subjects who carry the GNB3-825T allele show a reduced drive to breathing (minute ventilation) with an increase in inspiratory pCO ₂ and thus an increased CO ₂ tolerance. An increased CO ₂ tolerance represents an increased risk, in particular, for those people in whose working environment there may be a CO ₂ accumulation. The reduced CO ₂ sensitivity includes an increased risk of spontaneous loss of consciousness, e.g. B. in the context of the narcotic effect of CO ₂ . Affected are z. B. Professional divers, tunnel workers, pilots, submarine drivers, etc. With the detection of the GNB3-825T allele, such persons can be identified and warned so that the risk of accidents is reduced.

6. Prediction of osteoporosis

Generalized osteoporosis is one of the most common diseases of postmenopausal women and involves an increased risk of bone fractures. G proteins are significantly involved in processes that lead to bone remodeling. A changed activation of G-proteins is therefore significantly involved in the risk of developing osteoporosis (May, LG and Gay, CV Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts. J. Cell Biochem. 64 (1): 161- 170, 1997; Gordeladze, JO, Lund, HW, Jablonski, G., and Bruland, OS Diverse expression of G-proteins in human sarcoma cell lines with different osteogenic potential: Evidence for the involvement of G _i2 in cell proliferation. J. Cell. Biochem. 60: 95-106, 1996).

In this case too, carriers of the GNB3-825T allele show increased risk of developing osteoporosis.

7. Prediction of Alzheimer's disease

A change in the activation of G proteins and a change in the regulation of K ⁺ channels have been described in patients with Alzheimer's disease. A reduced activation of adenylyl cyclase after stimulation of β-adrenergic receptors has also been described. These phenomena can be attributed to an increased activatability of pertussis toxin-sensitive G proteins with expression of the Gß3 s splice variant (Okamoto, T., Takeda, S., Murayama, Y., Ogata, E., and Nishimoto, I. Ligand- dependent G protein coupling function of amyloid transmembrane precursor. J. Biol. Chem. 270: 4205-4208, 1995; Nishimoto, I., Okamoto, T., Matsuura, Y., Takahashi, S., Murayama, Y., and Ogata, E. Alzheimer amyloid protein precursor complexes with brain GTP-binding protein G _0. Nature 362: 75-79, 1993; Etcheberrigaray, R., Ito, E., Oka, K., Tofel-Grehl, B., Gibson , GE, and Alkon, DL Potassium channel dysfunction in fibroblasts identifies patients with Alzheimer's disease. Proc. Natl. Acad. Sci. USA 90: 8209-8213, 1993; Yamatsuji, T., Matsui, T., Okamoto, T., Komatsuzaki, K., Zakeda, S., Fukumoto, H., Iwatsubo, T., Suzuki, N., Asami-Odaka, A., Ireland, S., Kinane, TB, Giambarella, U., and Nishimoto, I G protein mediated neuronal DNA fragmentation induced b y familial Alzheimer's disease-associated mutants of APP. Science 272: 1349-1352, 1996; Cowburn, RF, Wiehager, B., Ravid, R., and Winblad, B. Acetylcholine muscarinic M2 receptor stimulated [ ³⁵ S] GTPγS binding shows regional selective changes in Alzheimer's disease postmortem brain. Neurodegeneration 5: 19-26, 1996).

As a result, carriers of the GNB3-825T allele have an increased Risk of developing Alzheimer's disease. In addition, the early in carriers of the GNB3-825T allele the onset of atherosclerosis described under 3 of Alzheimer's disease.

8. Predicting sexual dysfunction

Because of the tendency to increased atherosclerotic Vascular remodeling in carriers of the 825T allele increases in men the risk of erectile dysfunction and organic Impotence. This is due to the fact that the increased Atherosclerosis causes a decrease in erection required, increased inflow of blood into the erectile tissue of the penis. In women, the presence of an 825T Alleles in an analogous manner to reduced blood flow to the Sex organs (e.g. the clitoris) and thus one decreased sexual stimulability and orgasm ability to lead.

9. Prediction of thyroid dysfunction

Carriers of the 825T allele often have a disturbed Thyroid function on and must be using thyroid hormones (e.g. thyroxine).

10. Prediction of an increased risk of pregnancy

High pressure, edema formation and the so-called "HELLP syndrome" mean a serious risk to pregnancy, to both the life of the mother as well as for the life of the unborn Child. It has been found that in carriers of the 825T allele, who develop high pregnancy pressure (gestosis, Preeclampsia) a high risk of premature birth (birth before  the 37th week of pregnancy or the child's birth weight less than 2500 g), with additional for these children the risk of stillbirth or death after Childbirth exists. At the same time, that increases in women Carriers of the 825T allele are and under Gestational gestosis, the risk of spontaneous abortion (habitual abortions).

The detection of an 825T allele is therefore suitable, an elevated one Risk of death for the unborn child of pregnant women Predict gestational pregnancy.

Another object of the present invention is Pharmacogenetics of the GNB3-825T allele, i.e. H. the possibility, predict the effects of pharmaceuticals based on the genotype.

Most pharmaceuticals (hormones, receptor agonists) practice theirs Effect through receptors that couple to G proteins. Antagonists block hormone-receptor interaction.

It has now been found that genotyping at the GNB3- Lokus is suited to based on the effectiveness of pharmaceuticals to predict the genotype. This affects responsiveness in vivo for hormones, transmitters (also neurotransmitters) or Pharmaceuticals which activate such G protein heterotrimers which which contain G protein subunits Gβ3 and Gβ3s. In order to goes hand in hand with the prediction of reduced effectiveness of Hormones, neurotransmitters or pharmaceuticals that Stimulate protein subunit GαS, e.g. B. β-adrenergic Agonists. This also applies when using the Evidence of the Arg16Gly variant and the Gln27Glu variant in β2 adrenergic receptor. The presence of the GNB3-825T allele indicates a changed pharmacogenetics and should the specific selection of a form of therapy (pharmacological or non-pharmacological) and when dosing Drugs or hormones for hypertension, diabetes mellitus,  coronary artery disease, acute myocardial infarction with or without Irregular heartbeat, irregular heartbeat, Graft rejection etc. are taken into account.

In this context, the presence of the GNB3-825T- Alleles also predict the effectiveness of the administration of Erythropoietin on blood formation and prediction of onset hypertension under this therapy as well as the prediction of Danger under immunosuppressive therapy (e.g. with cyclosporin) to develop hypertension.

The effectiveness of Substances for therapy and prophylaxis of the migraine attack (Agonists at the serotonin receptor) can be predicted.

The following example demonstrates this. Here, an α2-adrenergic agonist (BHT 933) was applied intracoronary to test subjects, which leads to contraction of coronary vessels. This effect was quantified by the change in flow through these vessel sections. As shown in FIG. 8, an increased decrease in coronary blood flow is observed in carriers of the GNB3-825T allele, regardless of whether they are suffering from coronary artery disease (CAD). This means that in the case of carriers of the GNB3-825T allele, an increase in the action of such pharmaceuticals can be predicted.

Another object of the present invention is Manufacture of pharmaceuticals using the Gβ3s splice variant inhibit.

In principle, it is possible to synthesize chemicals that inhibit the function of the Gβ3s protein by z. B. lead to an accelerated degradation of the protein, or inhibit its interaction and combination with α and γ subunits of the human G protein. A screening system is required to identify such substances. For this purpose, the expression of the Gß3s subunit alone or in combination with different Gα and Gγ subunits in Sf9 insect cells, in other cells suitable for transfection, or the use of purified Gα and Gγβ3s subunits in a reconstitution system in Ab or Presence of a G protein-coupled receptor. Such systems can be used, for. B. Examine the effect of chemicals on the receptor-mediated binding of GTP to the Gα subunit, which can ultimately identify chemicals that inhibit the function of the Gβ3s subunit. In principle, such a test system can also be used as a "high-throughput screening system" for testing a large number of substances.

Appendix: Localization of the polymorphism in cDNA
Appendix 1: Original Levine β3 sequence

The exons are alternately underlined. The area that is eliminated by cryptic splicing is printed in bold.

Appendix 2: Sequence with the two polymorphisms (numbering according to the Levine sequence)

Claims (19)

1. Nucleic acid sequence coding for the Gβ3 subunit of human G protein with the sequence shown in Appendix 1. 2. Nucleic acid sequence coding for the Gβ3 subunit of human G protein with the sequence shown in Appendix 2. 3. Use of a gene change in the gene for the Gβ3- Subunit of the human G protein, at position 825 in Appendix 2 there is a substitution of cytosine by thymine and / or a substitution at position 1429 in Appendix 2 of cytosine through thymine to determine the risk disease associated with G protein misregistration fall ill. 4. Use according to claim 3 to determine the risk Type 2 diabetes mellitus, obesity and obesity, coronary artery Heart disease, myocardial infarction, sudden cardiac death, Osteoporosis, atherosclerosis, or neurodegenerative cerebrovascular diseases, especially Alzheimer's disease and / or an illness that is on an increased Immune system responsiveness depends on the disease. 5. Use of a gene change in the gene for the Gβ3- Subunit of the human G protein, at position 825 in Appendix 2 there is a substitution of cytosine by thymine and / or a substitution at position 1429 in Appendix 2 of cytosine through thymine to predict risk of women with cardiovascular disease, in particular High blood pressure or coronary artery disease and  Creation of a targeted hormone therapy around that reduce cardiovascular risk. 6. Procedure for determining the risk of a subject a disease associated with G protein misregistration in which the gene sequence for the Gβ3 subunit of human G protein of the test person with the gene sequence in Appendix 1 and / or Appendix 2 and in the event that they with the Gene sequence in Appendix 2 in position 825 and / or in position 1429 agrees, the subject an elevated Allocates disease risk. 7. The method according to claim 6, characterized in that for Determination of the risk of developing type 2 diabetes mellitus get sick, at the same time gene changes in the IRS1 gene (3931A- Variant; Gly971Arg), in the IRS2 gene, in the gene responsible for p85α regulatory subunit of the PI3 kinase encoded (1020 G → A; Codon 326 Met → Ile), in the gene for the β3-adrenergic Receptor encoded (Trp64Arg), in the gene for the β2-adrenergic Receptor coded (here in particular the Arg16Gly variant and the Gln27Glu variant), in the gene that is responsible for tumor necrosis factor α encodes and / or in the gene for leptin or the leptin receptor coded, examined. 8. The method according to claim 6, characterized in that for Determine the risk of becoming overweight and obese get sick, at the same time gene changes in the IRS1 gene (3931A- Variant; Gly971Arg), in the gene for the β3-adrenergic gene Receptor encoded (Trp64Arg variant) and / or in the gene for encodes the β2-adrenergic receptor (especially the Arg16Gly- Variant and the Gln27Glu variant) are examined. 9. The method according to claim 6, characterized in that for Determination of the risk of coronary artery disease and / or To get myocardial infarction, at the same time gene changes in IRS1 gene (3931A variant; Gly971Arg) are examined.   10. The method according to claim 6, characterized in that for Determination of the risk of diseases with a associated increased responsiveness of the immune system are to become ill, at the same time gene changes, in the β2- adrenergic receptor, (especially the Arg16Gly variant and the Gln27Glu variant) are examined. 11. Use of a gene change in the gene for the Gβ3 subunit of the human G protein, wherein there is a substitution of cytosine by thymine at position 825 in Appendix 2 and / or a substitution of cytosine by thymine at position 1429 in Appendix 2, for the identification of people with increased CO ₂ or hypoxia tolerance. 12. Use of a gene change in the gene for the Gβ3- Subunit of the human G protein, at position 825 in Appendix 2 there is a substitution of cytosine by thymine and / or a substitution at position 1429 in Appendix 2 of cytosine by thymine to predict the Responsiveness of a subject to hormones in vivo, Transmitters (also neurotransmitters) or pharmaceuticals, such Activate G protein heterotrimers, which the G protein Contain subunits Gβ3 and Gβ3s and / or the G protein Stimulate subunit GαS. 13. Use according to claim 12 for predicting the Responsiveness of a subject, at the same time the Arg16Gly variant and the Gln27Glu variant in the β2-adrenergic Receptor is detected. 14. Use according to claim 12 or 13 for specific Choosing a pharmacological or non-pharmacological Therapy form or the dosage of pharmaceuticals or hormones Hypertension, diabetes mellitus, coronary artery disease, acute  Myocardial infarction with or without irregular heartbeat, Cardiac arrhythmia and graft rejection. 15. Use according to claim 12 or 13, for predicting the Effectiveness of erythropoietin on blood formation and / or prediction of developing hypertension this therapy. 16. Use according to claim 12 or 13, for predicting the Danger under immunosuppressive therapy, especially with Cyclosporin to develop hypertension. 17. Use according to claim 12 or 13 for predicting the Effectiveness of substances for therapy and prophylaxis of the Migraine attack. 18. Use of a nucleic acid sequence that is complementary to is the nucleic acid sequence according to claim 1 or 2 for Manufacture of an antisense drug for therapy or Disease prevention. 19. Use of the human Gβ3s subunit protein G-protein in recombined systems or after transfection in suitable cell lines for the identification of chemicals, which inhibit the function of Gβ3s.